The proton-translocating NADH-quinone oxidoreductases are found in both mitochondria, where they are known as complex I, and in bacteria, where they are referred to as NDH-1. Both types of oxidoreductases are multi-subunit enzyme complexes localized in the respiratory chains of mitochondria and bacteria. These enzymes contain one FMN and at least 5 EPR-detectable iron-sulfur (FeS) clusters as cofactors. Investigation into the structure and function of the complex I/NDH-1 is important because (1) these complexes are the point of entry for the major fraction of electrons that traverse the respiratory chain, (2) these complexes translocate protons across the membranes allowing ATP synthesis to occur, (3) these complexes are believed to be the most elaborate of all the known FeS proteins, (4) the number of reports of human mitochondrial diseases involving defects at the level of this enzyme have increased. The overall goal of this grant application is to elucidate the structure and the mechanism of action of the proton-translocating NADH-quinone oxidoreductases in mitochondria and bacteria. In an effort to meet this objective we have cloned and sequenced the gene clusters encoding the Paracoccus and Thermus NDH-1 which are both composed of 14 dissimilar subunits and contain two segments, one located in the cytoplasmic space and the other in the membrane. The subunits bearing cofactors have been individually expressed in E. coli and characterized in this laboratory. In addition, the topology of the hydrophobic subunit has been determined. Furthermore, by use of a photoaffinity analogue of an inhibitor specific for the NDH-1/complex I, the inhibitor-binding subunit has been identified. The studies planned for this grant period are as follows: (i) Characterization of the remaining putative FeS cluster binding subunits and determination of the amino acid residues involved in coordination of the FeS clusters. (ii) Identification of the quinone-binding and specific inhibitor (pyridaben, fenpyroximate etc)-binding subunits as well as the amino acid residues involved in the ligation of the quinone and these inhibitors. (iii) Topological studies of the hydrophobic subunits in situ by immunochemical and cysteine mapping methods. (iv) Determination of the functional role of the intrinsic membrane segment of bovine heart complex I. (v) Determination of the effects of various culture conditions on the biosynthesis of the Paracoccus NDH-1 complex.